The rearing environment persistently modulates mouse phenotypes from the molecular to the behavioural level.

The phenotype of an organism results from its genotype and the influence of the environment throughout development. Even when using animals of the same genotype, independent studies may test animals of different phenotypes, resulting in poor replicability due to genotype-by-environment interactions. Thus, genetically defined strains of mice may respond differently to experimental treatments depending on their rearing environment. However, the extent of such phenotypic plasticity and its implications for the replicability of research findings have remained unknown. Here, we examined the extent to which common environmental differences between animal facilities modulate the phenotype of genetically homogeneous (inbred) mice. We conducted a comprehensive multicentre study, whereby inbred C57BL/6J mice from a single breeding cohort were allocated to and reared in 5 different animal facilities throughout early life and adolescence, before being transported to a single test laboratory. We found persistent effects of the rearing facility on the composition and heterogeneity of the gut microbial community. These effects were paralleled by persistent differences in body weight and in the behavioural phenotype of the mice. Furthermore, we show that environmental variation among animal facilities is strong enough to influence epigenetic patterns in neurons at the level of chromatin organisation. We detected changes in chromatin organisation in the regulatory regions of genes involved in nucleosome assembly, neuronal differentiation, synaptic plasticity, and regulation of behaviour. Our findings demonstrate that common environmental differences between animal facilities may produce facility-specific phenotypes, from the molecular to the behavioural level. Furthermore, they highlight an important limitation of inferences from single-laboratory studies and thus argue that study designs should take environmental background into account to increase the robustness and replicability of findings.

Postoperative Severity Assessment in Sheep.

INTRODUCTION: Sheep are frequently used in translational surgical orthopedic studies. Naturally, a good pain management is mandatory for animal welfare, although it is also important with regard to data quality. However, methods for adequate severity assessment, especially considering pain, are rather rare regarding large animal models. Therefore, in the present study, accompanying a surgical pilot study, telemetry and the Sheep Grimace Scale (SGS) were used in addition to clinical scoring for severity assessment after surgical interventions in sheep. METHODS: Telemetric devices were implanted in a first surgery subcutaneously into four German black-headed mutton ewes (4-5 years, 77-115 kg). After 3-4 weeks of recovery, sheep underwent tendon ablation of the left M. infraspinatus. Clinical scoring and video recordings for SGS analysis were performed after both surgeries, and the heart rate (HR) and general activity were monitored by telemetry. RESULTS: Immediately after surgery, clinical score and HR were slightly increased, and activity was decreased in individual sheep after both surgeries. The SGS mildly elevated directly after transmitter implantation but increased to higher levels after tendon ablation immediately after surgery and on the following day. CONCLUSION: In summary, SGS- and telemetry-derived data were suitable to detect postoperative pain in sheep with the potential to improve individual pain recognition and postoperative management, which consequently contributes to refinement.

The Rise of Three Rs Centres and Platforms in Europe.

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term 'the Three Rs', which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro, in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years - not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA's 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as 'on the ground' facilitators of Directive 2010/63/EU. They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.

The Current Status and Work of Three Rs Centres and Platforms in Europe.

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

Running in the wheel: Defining individual severity levels in mice.

The fine-scale grading of the severity experienced by animals used in research constitutes a key element of the 3Rs (replace, reduce, and refine) principles and a legal requirement in the European Union Directive 2010/63/EU. Particularly, the exact assessment of all signs of pain, suffering, and distress experienced by laboratory animals represents a prerequisite to develop refinement strategies. However, minimal and noninvasive methods for an evidence-based severity assessment are scarce. Therefore, we investigated whether voluntary wheel running (VWR) provides an observer-independent behaviour-centred approach to grade severity experienced by C57BL/6J mice undergoing various treatments. In a mouse model of chemically induced acute colitis, VWR behaviour was directly related to colitis severity, whereas clinical scoring did not sensitively reflect severity but rather indicated marginal signs of compromised welfare. Unsupervised k-means algorithm-based cluster analysis of body weight and VWR data enabled the discrimination of cluster borders and distinct levels of severity. The validity of the cluster analysis was affirmed in a mouse model of acute restraint stress. This method was also applicable to uncover and grade the impact of serial blood sampling on the animal's welfare, underlined by increased histological scores in the colitis model. To reflect the entirety of severity in a multidimensional model, the presented approach may have to be calibrated and validated in other animal models requiring the integration of further parameters. In this experimental set up, however, the automated assessment of an emotional/motivational driven behaviour and subsequent integration of the data into a mathematical model enabled unbiased individual severity grading in laboratory mice, thereby providing an essential contribution to the 3Rs principles.

CD14 Plays a Protective Role in Experimental Inflammatory Bowel Disease by Enhancing Intestinal Barrier Function.

Intestinal homeostasis disturbance through intestinal barrier disruption presumably plays a key role in inflammatory bowel disease (IBD) development. Genetic and candidate gene analyses in an Il10-deficient IBD mouse model system identified Cd14 as a potentially protective candidate gene. The role of Cd14 in colitis development was determined using dextran sulfate sodium (DSS)-induced acute and an Il10-deficiency-induced chronic model of intestinal inflammation. Intestinal permeability was investigated by fluorescein isothiocyanate-dextran uptake assay, quantitative RT-PCR analysis of tight junction proteins, myosin light chain kinase, and proinflammatory cytokine expression. Immunohistological staining of occludin, Ki-67, NF-κB-p65, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay was performed, and intestinal inflammation severity was evaluated histologically. Untreated B6-Cd14-/- mice and wild-type controls did not differ in intestinal barrier function. However, DSS-treated Cd14-deficient and B6-Il10-/-Cd14-/- mice exhibited more severe intestinal barrier disruption, with increased histological scores and proinflammatory cytokine expression, compared to controls. Therefore, Cd14 deficiency did not influence epithelial integrity under steady-state conditions but caused intestinal barrier dysfunction under inflammation. As expected, CD14 overexpression increased barrier integrity. No difference in intestinal epithelial NF-κB translocation was observed between the investigated groups. Intestinal myosin light chain kinase expression decreased in Cd14-deficient mice under steady-state conditions and in the chronic model, whereas no difference was detected in the DSS models. Thus, CD14 plays a protective role in IBD development by enhancing intestinal barrier function.

Alterations in anthropometric, inflammatory and mental health parameters during Ramadan intermittent fasting in a group of healthy people: a prospective cohort study.

Fasting has been practiced with different time span in different areas of the world and for various reasons. One of the types of fasting regimens is Ramadan intermittent fasting (RIF), which is described as intermittent dry fasting and known as the most commonly practiced form of religious fasting. Different studies have shown its effects on body composition parameters and mental health, fatigue and quality of life (QoL). Elucidating the relationship of RIF on biological parameters would also be of importance to show its mechanism. Therefore, we evaluated several biological mediators related to mental health, such as ß-nerve growth factor (ß-NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and insulin-like growth factor-1 (IGF-1), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and matrix-metalloproteinase-9 (MMP-9). This study consisted of fasting (FG; n = 25) and non-fasting group (NFG; n = 25). Four different time points were assessed for FG: one week before (T1), mid (T2), last days (T3), and one week after (T4) RIF. T1 and T3 were the assessment time points for NFG. Biological mediators were determined from serum samples by using Human Magnetic Luminex and enzyme-linked immunosorbent assay. Furthermore, we then performed correlation analyses between biological mediators and our previously published clinical parameters including body composition and mental health parameters at all time points. Significant alterations were shown in FG for ß-NGF (T2vsT3, p < 0.05; T2vsT4, p < 0.05), GDNF (T1vsT4, p < 0.05; T2vsT4, p < 0.05), IL-8 (T2vsT3, p < 0.05; T3vsT4, p < 0.05), TNF-α (T1vsT3, p < 0.05; T1vsT4, p < 0.001; T2vsT4, p < 0.001), and MMP-9 (T1vsT4, p < 0.01). There were no statistically significant differences between FG and NFG in all biological mediators at T1 and T3. Correlation analysis showed that MMP-9 levels had negative correlation with body mass index (BMI) at T3. At T3 BDNF levels had negative correlation with Epworth Sleepiness Scale (ESS) as one of measured QoL parameters. ß-NGF, GDNF, TNF-α, and MMP-9 had positive correlation with some of body composition and mental health parameters. Findings demonstrate that RIF altered different biological mediators could give benefit to health. Its benefit is mediated by the alteration of biological mediators.

Mapping colitis susceptibility in mouse models: distal chromosome 3 contains major loci related to Cdcs1.

Inflammatory bowel disease (IBD) summarizes a group of chronic intestinal disorders with Crohn's disease and ulcerative colitis being most prominent. Though much effort is put into identification of causative factors, its etiology is still not understood. Risk factors for disease development include genetic predisposition and environmental triggers. Crucial for identification and analysis of relevant factors are mouse models. Experimental IBD in mice occurs spontaneously or is induced by chemicals, cell transfer, pathogens, or genetic mutation. These models were utilized for analyzing genetic contribution to disease and genotype-environmental interactions. In these studies, a variety of modifier loci were identified, thereby demonstrating the complexity of disease. A major contribution of distal chromosome 3 was independently replicated in several studies. The first colitogenic QTL in this region was detected using the IL-10-deficient mouse model and called cytokine deficiency-induced colitis susceptibility (Cdcs)1. This quantitative trait locus contains at least three subintervals with independent genetic factors. This locus or defined subintervals were replicated in at least seven studies, using models based on dysregulation of innate or adaptive immunity or pathogen control. In this review we illustrate the various models used for genetic mapping of susceptibility to experimental IBD and display Cdcs1-related loci as well as the mechanism of their contribution identified so far.

Stromal cell heterogeneity in lymphoid organs.

Although the role of stromal cells has not been clearly defined, these cells have been described as forming the extracellular matrix in all lymphoid organs. Their important role in facilitating the development of immune cells in the thymus and bone marrow has long been known. In contrast, stromal cells have been found in secondary lymphoid organs and it has been shown that they are important mediators during organogenesis. More recently, their important function in the guidance and survival of immune cells has been documented. Here, we describe the important role of stromal cells within secondary lymphoid organs and highlight the fact that the immunological function of stromal cells is site-specific and unique in each lymphoid organ.

Grimace scale assessment during Citrobacter rodentium inflammation and colitis development in laboratory mice.

Introduction: Bacterial infections and chronic intestinal inflammations triggered by genetic susceptibility, environment or an imbalance in the intestinal microbiome are usually long-lasting and painful diseases in which the development and maintenance of these various intestinal inflammations is not yet fully understood, research is still needed. This still requires the use of animal models and is subject to the refinement principle of the 3Rs, to minimize suffering or pain perceived by the animals. With regard to this, the present study aimed at the recognition of pain using the mouse grimace scale (MGS) during chronic intestinal colitis due to dextran sodium sulfate (DSS) treatment or after infection with Citrobacter rodentium. Methods: In this study 56 animals were included which were divided into 2 experimental groups: 1. chronic intestinal inflammation (n = 9) and 2. acute intestinal inflammation (with (n = 23) and without (n = 24) C. rodentium infection). Before the induction of intestinal inflammation in one of the animal models, mice underwent an abdominal surgery and the live MGS from the cage side and a clinical score were assessed before (bsl) and after 2, 4, 6, 8, 24, and 48 hours. Results: The highest clinical score as well as the highest live MGS was detected 2 hours after surgery and almost no sign of pain or severity were detected after 24 and 48 hours. Eight weeks after abdominal surgery B6-Il4/Il10-/- mice were treated with DSS to trigger chronic intestinal colitis. During the acute phase as well as the chronic phase of the experiment, the live MGS and a clinical score were evaluated. The clinical score increased after DSS administration due to weight loss of the animals but no change of the live MGS was observed. In the second C57BL/6J mouse model, after infection with C. rodentium the clinical score increased but again, no increased score values in the live MGS was detectable. Discussion: In conclusion, the live MGS detected post-operative pain, but indicated no pain during DSS-induced colitis or C. rodentium infection. In contrast, clinical scoring and here especially the weight loss revealed a decreased wellbeing due to surgery and intestinal inflammation.

Mimicking acute airway tissue damage using femtosecond laser nanosurgery in airway organoids.

Airway organoids derived from adult murine epithelial cells represent a complex 3D in vitro system mimicking the airway epithelial tissue's native cell composition and physiological properties. In combination with a precise damage induction via femtosecond laser-based nanosurgery, this model might allow for the examination of intra- and intercellular dynamics in the course of repair processes with a high spatio-temporal resolution, which can hardly be reached using in vivo approaches. For characterization of the organoids' response to single or multiple-cell ablation, we first analyzed overall organoid survival and found that airway organoids were capable of efficiently repairing damage induced by femtosecond laser-based ablation of a single to ten cells within 24 h. An EdU staining assay further revealed a steady proliferative potential of airway organoid cells. Especially in the case of ablation of five cells, proliferation was enhanced within the first 4 h upon damage induction, whereas ablation of ten cells was followed by a slight decrease in proliferation within this time frame. Analyzing individual trajectories of single cells within airway organoids, we found an increased migratory behavior in cells within close proximity to the ablation site following the ablation of ten, but not five cells. Bulk RNA sequencing and subsequent enrichment analysis revealed the differential expression of sets of genes involved in the regulation of epithelial repair, distinct signaling pathway activities such as Notch signaling, as well as cell migration after laser-based ablation. Together, our findings demonstrate that organoid repair upon ablation of ten cells involves key processes by which native airway epithelial wound healing is regulated. This marks the herein presented in vitro damage model suitable to study repair processes following localized airway injury, thereby posing a novel approach to gain insights into the mechanisms driving epithelial repair on a single-cell level.

Epithelial restitution in 3D - Revealing biomechanical and physiochemical dynamics in intestinal organoids via fs laser nanosurgery.

Intestinal organoids represent a three-dimensional cell culture system mimicking the mammalian intestine. The application of single-cell ablation for defined wounding via a femtosecond laser system within the crypt base allowed us to study cell dynamics during epithelial restitution. Neighboring cells formed a contractile actin ring encircling the damaged cell, changed the cellular aspect ratio, and immediately closed the barrier. Using traction force microscopy, we observed major forces at the ablation site and additional forces on the crypt sides. Inhibitors of the actomyosin-based mobility of the cells led to the failure of restoring the barrier. Close to the ablation site, high-frequency calcium flickering and propagation of calcium waves occured that synchronized with the contraction of the epithelial layer. We observed an increased signal and nuclear translocation of YAP-1. In conclusion, our approach enabled, for the first time, to unveil the intricacies of epithelial restitution beyond in vivo models by employing precise laser-induced damage in colonoids.

Lymph node stromal cells strongly influence immune response suppression.

Many pathogens are initially encountered in the gut, where the decision is made to mount an immune response or induce tolerance. The mesentric lymph node (mLN) has been shown to be involved in immune response and much more in oral tolerance induction. Furthermore, using an in vivo transplantation model, we showed recently that lymph node (LN) stromal cells can affect T-cell function and influence the IgA response by supporting a site-specific environment. To elucidate the importance of LN stromal cells for tolerance induction, mLN or peripheral LN were transplanted into mice (mLNtx or pLNtx) and oral tolerance was induced via ovalbumin. A reduced delayed-type hypersensitivity (DTH) response was detected in pLNtx compared to mLNtx mice. Reduced IL-10 expression, reduced percentages of Tregs, and increased proportions of B cells were identified within the pLNtx. The increase of B cells resulted in a specific immunoglobulin production undetectable in mLNtx. Moreover, transferred IgG(+) cells of tolerized peripheral LN induced a strong reduction of the delayed-type hypersensitivity response, whereas CD4(+) cells were less efficient. Thus, stromal cells have a high impact on creating a unique environment. Furthermore, the environment of pLNtx induces a tolerogenic phenotype by B-cell accumulation and antibody production.

Diet-induced obesity results in impaired oral tolerance induction.

INTRODUCTION: Obesity increases the risk of several diseases, such as type 2 diabetes mellitus and cardiovascular disease. Obesity also affects the immune system. When dietary lipids are transported via the lymphatics, they pass the mesenteric lymph nodes (mLNs). In these secondary lymphoid organs, immune responses towards pathogens are generated, or tolerance against harmless antigens is induced. METHODS: In this study, the effects of diet-induced obesity (DIO) on mLN induced oral tolerance induction were examined in C57BL/6NCrl mice. Therefore, mice were fed a high-fat or a low-fat diet for 14 weeks. After 10 weeks of feeding oral tolerance induction started, ending up in measuring the delayed-type hypersensitivity reaction, the cell subset composition and cytokine expression. RESULTS: We detected an impaired oral tolerance induction during DIO, but changes were reversible after switching the feed to standard chow. Thus, the altered immunological function of mLNs depends on the intake of dietary lipids. Additionally, our results show an influence of the microenvironment on the development of oral tolerance during DIO as oral tolerance was induced in transplanted peripheral lymph nodes. CONCLUSION: This indicates a functional influence of dietary lipids on stromal cells involved in immune system induction in the mLNs.

Investigation of Colonic Regeneration via Precise Damage Application Using Femtosecond Laser-Based Nanosurgery.

Organoids represent the cellular composition of natural tissue. So called colonoids, organoids derived from colon tissue, are a good model for understanding regeneration. However, next to the cellular composition, the surrounding matrix, the cell-cell interactions, and environmental factors have to be considered. This requires new approaches for the manipulation of a colonoid. Of key interest is the precise application of localized damage and the following cellular reaction. We have established multiphoton imaging in combination with femtosecond laser-based cellular nanosurgery in colonoids to ablate single cells in the colonoids' crypts, the proliferative zones, and the differentiated zones. We observed that half of the colonoids recovered within six hours after manipulation. An invagination of the damaged cell and closing of the structure was observed. In about a third of the cases of targeted crypt damage, it caused a stop in crypt proliferation. In the majority of colonoids ablated in the crypt, the damage led to an increase in Wnt signalling, indicated via a fluorescent lentiviral biosensor. qRT-PCR analysis showed increased expression of various proliferation and Wnt-associated genes in response to damage. Our new model of probing colonoid regeneration paves the way to better understand organoid dynamics on a single cell level.

Acetyl-CoA-Carboxylase 1-mediated de novo fatty acid synthesis sustains Lgr5+ intestinal stem cell function.

Basic processes of the fatty acid metabolism have an important impact on the function of intestinal epithelial cells (IEC). However, while the role of cellular fatty acid oxidation is well appreciated, it is not clear how de novo fatty acid synthesis (FAS) influences the biology of IECs. We report here that interfering with de novo FAS by deletion of the enzyme Acetyl-CoA-Carboxylase (ACC)1 in IECs results in the loss of epithelial crypt structures and a specific decline in Lgr5+ intestinal epithelial stem cells (ISC). Mechanistically, ACC1-mediated de novo FAS supports the formation of intestinal organoids and the differentiation of complex crypt structures by sustaining the nuclear accumulation of PPARδ/ß-catenin in ISCs. The dependency of ISCs on cellular de novo FAS is tuned by the availability of environmental lipids, as an excess delivery of external fatty acids is sufficient to rescue the defect in crypt formation. Finally, inhibition of ACC1 reduces the formation of tumors in colitis-associated colon cancer, together highlighting the importance of cellular lipogenesis for sustaining ISC function and providing a potential perspective to colon cancer therapy.

Stromal cells directly mediate the re-establishment of the lymph node compartments after transplantation by CXCR5 or CCL19/21 signalling.

Lymph nodes (LN) are highly organized and have characteristic compartments. Destruction of these compartments leads to an inability to fulfil their immunological function. However, it is not yet clearly understood which mechanisms are involved in the development and maintenance of this organization. After transplantation of LN into the mesentery, the LN regenerate to fully functional LN. In this study, the question was addressed, how stromal cells in the B-cell follicles (follicular dendritic cells), which were identified by CD21/CD35, and stromal cells in the T-cell area (gp38+ cells) are involved via chemokine signalling. The gp38+ cells and CD21/CD35+ cells were detected in the transplanted LN (EGFP, plt/plt and CXCR5(-/-) mice) over a period of 8 weeks to analyse their competence to reconstruct the compartmental organization. The presence of gp38+ cells was stable during regeneration and these cells reconstructed the T-cell area within 4 weeks. After transplantation of plt/plt LN CCL19/CCL21 expression was observed leading to partial restoration of the T-cell area. In contrast, there were changes in the presence and morphology of CD21/CD35+ cells within the B-cell area during reconstruction, which was dependent on the presence of B cells and CXCL13/CXCR5 signalling. Hence, CD21/CD35+ cells and gp38+ cells are involved in the establishment of the compartmental organization of lymph nodes but using different ways to recruit lymphocytes via chemokine signalling.

Lymph node transplantation and its immunological significance in animal models.

Lymph nodes (LNs) are distributed all over the body and whatever the site consists of the same cell populations. However, there are great differences between LN from different draining areas. For example, in mesenteric LN, homing molecules, for example, CCR9 and α4ß7 integrin, were induced and cytokines, for example, IL-4, were produced on higher levels compared to peripheral LN. To study the immunological functions of LN, LN transplantation was performed in some specific areas using different animal models. Many groups investigated not only the regeneration of transplanted LN but also the induction of immune responses or tolerance after transplantation. Existing differences between LNs were still detectable after transplantation. Most important, stromal cells of the LN were identified as responsible for these differences. They survive during regeneration and were shown to reconstruct not only the structure of the new LN but also the microenvironment.

Ramadan Fasting in Germany (17-18 h/Day): Effect on Cortisol and Brain-Derived Neurotrophic Factor in Association With Mood and Body Composition Parameters.

Ramadan fasting (RF) is a type of diurnal intermittent fasting. Previous studies reported the benefits of RF in healthy subjects on mood and health related to quality of life (QoL). Cortisol and brain-derived neurotrophic factor (BDNF) have been shown to play a role in mood, body composition parameters, and health-related QoL. This study aimed at elucidating the mechanism of the benefit of RF, particularly cortisol and BNDF and their association with mood and QoL. Insulin growth factor-1 (IGF-1), interleukin (IL)-8, matrix metalloproteinase (MMP)-9, and myoglobin were determined. Thirty-four healthy men and women were recruited. Serum from peripheral venous blood samples was collected at five time points: 1 week before RF (T1); mid of RF (T2), last days of RF (T3), 1 week after RF (T4), and 1 month after RF (T5). The amounts of biological mediators in the serum samples were determined by enzyme-linked immunosorbent assay (ELISA) and Luminex assays. BDNF and cortisol significantly decreased at T3 (p < 0.05) and T4 (p < 0.001) compared to T1, respectively. It seems the benefits of RF for mood-related symptoms are mediated by different biological mediators, particularly cortisol and BDNF.

Intestinal Organoids in Colitis Research: Focusing on Variability and Cryopreservation.

In recent years, stem cell-derived organoids have become a cell culture standard that is widely used for studying various scientific issues that were previously investigated through animal experiments and using common tumor cell lines. After their initial hype, concerns regarding their standardization have been raised. Here, we aim to provide some insights into our experience in standardizing murine colonic epithelial organoids, which we use as a replacement method for research on inflammatory bowel disease. Considering good scientific practice, we examined various factors that might challenge the design and outcome of experiments using these organoids. First, to analyze the impact of antibiotics/antimycotics, we performed kinetic experiments using ZellShield® and measured the gene expression levels of the tight junction markers Ocln, Zo-1, and Cldn4, the proliferation marker Ki67, and the proinflammatory cytokine Tnfα. Because we found no differences between cultivations with and without ZellShield®, we then performed infection experiments using the probiotic Escherichia coli Nissle 1917 as an already established model setup to analyze the impact of technical, interexperimental, and biologic replicates. We demonstrate that interexperimental differences pose the greatest challenge for reproducibility and explain our strategies for addressing these differences. Additionally, we conducted infection experiments using freshly isolated and cryopreserved/thawed organoids and found that cryopreservation influenced the experimental outcome during early passages. Formerly cryopreserved colonoids exhibited a premature appearance and a higher proinflammatory response to bacterial stimulation. Therefore, we recommend analyzing the growth characteristics and reliability of cryopreserved organoids before to their use in experiments together with conducting several independent experiments under standardized conditions. Taken together, our findings demonstrate that organoid culture, if standardized, constitutes a good tool for reducing the need for animal experiments and might further improve our understanding of, for example, the role of epithelial cells in inflammatory bowel disease development.